Abscisic acid is a central stress hormone in plants which mediates rapid cellular responses, down regulates cell proliferation, causes cell cycle arrest and inhibits growth. Signal transduction mechanisms that down regulate cell proliferation are of central importance for controlling mitogenesis. Abscisic acid (ABA) signal transduction will be investigated in Arabidopsis guard cells, which have been developed as a powerful molecular genetic, genomic and time-resolved signaling system for quantitatively dissecting the complex molecular machinery mediating specificity in early signaling cascades. We will analyze the cellular mechanisms of a newly revealed major early ABA signal transduction pathway. In brief, we showed that ABA causes rapid production of reactive oxygen species (ROS) in guard cells, which in turn activate plasma membrane calcium (Ca2+) channels. This produces a defined window of Ca2+ oscillation parameters that are required for ABA signal transduction. We identified ABA signaling mutants that function at distinct points in the newly recognized ABA-ROS-Ca2+. oscillation signaling pathway, including mutations in the NADPH oxidase genes AtrbohD &F, PP2C phosphatases and the mRNA cap binding protein ABH1. We will test the hypotheses: that ABA biochemically activates membrane NADPH oxidases causing ROS production; that negatively regulating Ca2+ sensors function in reading specific Ca2+ oscillations; and that abh1 amplifies ABA-induced Ca2+ oscillations due to mRNA processing of early ABA signal transducers. To address these hypotheses, we will pursue the following specific aims: (1) Characterize biochemical and molecular mechanisms mediating rapid ABA-induced ROS production and characterize proteins complexed with the abi1-1 PP2C. (2) Analyze mechanisms by which ROS activate Ca2+ channels and isolate the Ca2+ channel genes. (3) The mechanisms responsible for reading specific calcium oscillations remain largely unknown in eukaryotes. Characterize the roles of the TCH2 and CalcineurinB-AtCBL Ca2+ sensory proteins in reading Ca2+ oscillations. (4) Characterize the mechanisms by which the abh1 mRNA cap binding mutant amplifies ABA-induced Ca2+ oscillations. A new virus-induced gene silencing-based screen will be pursued for identifying novel redundant ABA signaling mutants. These studies will reveal important fundamental mechanisms by which a newly recognized early signaling pathway controls ABA stress signal transduction and will contribute to a general understanding of Ca2+ signaling specificity in eukaryotes.